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Aseptic techniques
- 1. Renjimol Raveendran Junior ResearchFellow-DBT Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Thiruvananthapuram
- 2. INTRODUCTION • Asepsis: Stateof being free from microorganisms. • Aseptic technique means using practices and procedures to prevent contamination from pathogens. • The labware or culture medium, plant material or environment in the laboratory, instruments used for culture or the operator himself, all are sources of infection. • The tissue culture medium being rich in sugar and other organic and inorganic nutrients supports good growth of microorganisms, such as fungi and bacteria.
- 3. • These microorganismsmay grow faster than the plant tissues and may also secrete toxic wastes that inhibiting growth of cultured tissues, finally killing them. HISTORY • The aseptic practices were started in the mid- 19th century by Louis Pateur to disprove the spontaneous generation with the concepts of germ theory. • Antiseptic practices were introduced by Joseph Lister by using Phenol as disinfectant in 1860s. • In 1890 William Stewart Halstead requested that the Goodyear Rubber Company make a pair of thin rubber gloves
- 4. STERILIZATION • Sterilization: Theprocess of making something free from bacteria or living organisms.
- 5. Moist Heat Sterilization Autoclave;(a). Horizontal (b). Vertical • Heat under high pressure is used (Autoclave, pressure cooker, etc.)
- 6. Dry Heat Sterilization •Removing all microorganisms with longer exposure time (1.5 to 3 hours) and higher temperatures than moist heat sterilization. Spirit Lamp
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- 10. MAINTENANCE OF ASEPTICCONDITIONS Glassware and Plastic-ware: • For pre-sterilized medium the culture vials with proper closure may be sterilized by autoclaving or dry heating in an oven at 160–180 C for 3 h. • Only polypropylene, polymethylpentene, polyallomer, Tefzel ETFE and Teflon FEP may be repeatedly autoclaved at 121 C. • Polystyrene, polyvinyl chlorides, styrene acrylonitrile, are not autoclavable at all. • Culture Medium: the culture medium is sterilized by autoclaving at 15 psi and 121 C for 15–40 min.
- 11. • Vitamines, Hormones& other thermolabile components can be filter sterilized. • Instruments: sterilized before use by wrapping in aluminium foil and autoclaved followed by sterilized several times by dipping in 95 % ethanol and flaming and used after cooling. • Plant Material: Surface sterilization. • Transfer Area : LAF (HEPA filters-0.3µm), Spirit lamp, UV germicidal lamp, etc. • Growth room: Fumigation (mixture of 75 g potassium permanganate and 35 ml of 40 % formalin), cleaning with germicidal solutions such as ethyl alcohol, UV lamps, Regular house-keeping and monitoring spore count.
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